Cholesterol (Compound 1) is a critical constituent of membranes of animal cells. Cells acquire exogenous forms of this sterol through multiple mechanisms involving structurally distinct cell surface receptors. Lipoprotein particles such as low-density lipoprotein (LDL) and high-density lipoprotein (HDL) carry cholesteryl esters (Compound 2) and associated protein and lipid components throughout the bloodstream. Cells expressing LDL and HDL receptors actively internalize these natural nanoparticles via receptor-mediated endocytosis. In contrast, cellular uptake of free (unesterified) cholesterol, found in mixed micelles, involves the direct binding of this sterol to other receptors, such as Niemann-Pick C1-like protein (NPC1L1) on cell surfaces. This receptor for free cholesterol was identified as a target of ezetimibe (Compound 3), a drug used to treat hypercholesterolemia. Recent studies suggest that although NPC1L1 is a primary target of ezetimibe and its glucuronide metabolite, other proteins such as the HDL receptor SR-BI can also be inhibited by this drug. Also, there may be other mechanisms for cholesterol, cholesteryl ester, ezetimibe, or other agent to enter into cells, and such mechanisms may be exploited for delivery of agents (e.g., diagnostic, therapeutic, imaging, or other) into cells.

Some previously identified derivatives of cholesterol have numerous important biological applications. These known compounds, particularly cholesteryl carbamates, have been used to facilitate the delivery of small inhibitory RNA (siRNA), enhance DNA transfection, probe cellular membrane subdomains, and have been proposed for tumor targeting applications. For example, cellular uptake of cholesteryl carbamate-conjugated siRNA in vitro and in vivo is similar to uptake of cholesteryl esters, requiring binding to HDL or LDL, followed by internalization via HDL or LDL receptors. This initial lipoprotein-binding step presumably slows uptake as compared to internalization of free cholesterol via direct binding to cell surface receptors. Because cholesteryl carbamate-linked siRNAs are only internalized after binding lipoproteins, the presence of high concentrations of serum in media typically used for cell culture (e.g. 10%) substantially reduces the activity of these compounds, likely because of competition between serum lipoproteins and cognate cell surface receptors.
Therefore, there is a need for continued development of cholesterol derivatives for increasing the binding of cells and efficacy of delivery of agents into cells.